Prepared foods containing triglyceride-recrystallized non-esterified phytosterols

ABSTRACT

A dietary supplement is provided which includes at least one triglyceride-based edible fat and between 3% and 50% by weight of triglyceride recrystallized phytosterols. The dietary supplement can be in the form of a capsule, pill or wafer. Additionally, the dietary supplement can be combined with protein, vitamins, minerals, or combinations thereof.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/475,575, filed Jun. 26, 2006, which in turn is a continuation of U.S.application Ser. No. 10/677,634, filed Oct. 1, 2003, which is acontinuation-in-part of PCT Application PCT/US02/36809, filed Nov. 14,2002, and a continuation-in-part of U.S. application Ser. No.10/295,929, filed Nov. 14, 2002, which claim the benefit of U.S.Provisional Application No. 60/332,434, filed Nov. 16, 2001, each ofwhich is incorporated herein by reference in its entirety, including allfigures and tables.

BACKGROUND OF THE INVENTION

The present invention relates to prepared foods, such as fried snackfoods, fortified with non-esterified phytosterols delivered in fats oroils that are essentially free of emulsifiers and the like, and to theutility of such phytosterols for stabilizing heated fats and oilsagainst oxidation, as well as to the surprising bioavailability oftriglyceride-recrystallized phytosterols in such foods, for decreasingplasma cholesterol levels in mammals.

It has been a widely held belief that to obtain appreciable benefit fromphytosterols, i.e., either plant sterols, stanols, or combinationsthereof [including beta-sitosterol, beta-sitostanol, campesterol,campestanol, stigmasterol, stigmastanol, brassicasterol, brassicastanol,clionasterol and clionastanol (collectively termed phytosterol orphytosterols)] for lowering plasma cholesterol, the phytosterol shouldbe dissolved in an edible oil or other solvent so that it can entermicelles in the small intestine to inhibit the absorption ofcholesterol.

This belief has been supported by early research carried out in the1950s through the 1970s showing that large doses of phytosterols intheir solid form, i.e., coarse powders, were required to achievemeaningful decreases in plasma cholesterol levels. For example, in 1956,Faquhar et al., (Circulation, 14, 77-82, 1956) showed that doses of12-18 g per day of beta sitosterol (provided in divided doses) wererequired to achieve a 15-20% lowering of serum cholesterol in males withatherosclerosis. In another study, 9 g per day (3 g t.i.d.) ofsoybean-derived phytosterols were required to lower plasma cholesterolapproximately 9% (Kucchodkar et al., Atherosclerosis, 23, 239-248,1976). In yet another study, 3-9 g per day of tall oil-derivedphytosterols were required to lower plasma cholesterol approximately 12%(Lees et al., Atherosclerosis, 28: 325-333, 1977). In a recent study,1.7 g per day of finely powdered tall oil-derived phytosterols weresufficient to lower total plasma cholesterol by 9% and LDL-cholesterolby about 15% (Jones et al., Am J Clin Nutr, 69: 1144-1150, 1999).

It has been generally appreciated that phytosterols such as alpha andbeta sitosterol, stigmasterol, campesterol, and the correspondingsaturated (chemically reduced or hydrogenated) “stanol” species, areinsoluble in water, and only slightly soluble in edible oils.Accordingly, to promote the solubilization of phytosterols, and theirefficacy in lowering plasma cholesterol, U.S. Pat. No. 6,025,348 by Gotoet al. describes the incorporation of at least 15% and as much as 70% byweight or more of a polyhydric alcohol/fatty acid ester (includingglycerol fatty acid esters containing at least two esterified and atleast one unesterified hydroxyl group such as diacylglycerols ordiglycerides), into a fat. Between 1.2% and 4.7% by weight ofphytosterol is incorporated into the polyhydric alcohol/fatty acid estercontaining fat composition.

U.S. Pat. No. 6.139,897 by Goto et al. describes an oil or fatcomposition containing 80% or more diacylglycerol and up to 20%phytosterol. The high proportion of diacylglycerol assures solubility ordispersal of the phytosterol to provide a cholesterol-lowering fatsubstitute.

U.S. Pat. No. 5998,396 by Nakano et al., describes an edible oilcontaining a phytosterol, vitamin E, and an emulsifier rendering thephytosterol soluble in both the vitamin E and the edible oil.

U.S. Pat. No. 5,419,925 by Seiden et al. describes a reduced calorie fatcomposition based upon a substantially non-digestible polyol fatty acidpolyester plus reduced calorie medium chain triglycerides and otherreduced calorie fats or noncaloric fat replacements including plantsterol esters that are soluble in such fat compositions. Free fattyacids, vitamin E and tocotrienol have each been utilized by otherinventors to promote the solubilization of phytosterols in fats andoils, with the expectation that the cholesterol lowering properties ofvarious phytosterols would be improved.

U.S. Pat. No. 5,244,887 by Straub describes the preparation of acholesterol-lowering food additive composition with plant stanols,including: (i) an edible carrier such as an oil, monoglyceride,diglyceride, triglyceride, tocopherol, alcohol or polyol, (ii) anantioxidant and (iii) a dispersant or detergent-like material such aslecithin, or other phospholipids, sodium lauryl sulfate, a fatty acid,salts of fatty acids, or a fatty acid ester. Straub cites researchshowing that 1.5 grams per day of a stanol mixture derived from soybeansterols lowered blood cholesterol by 15% after 4 weeks of therapy, andbelieves that these stanols are preferred to sterols based upon lessstanol absorption from the G.I. tract and better heat stability in airthan sterols.

U.S. Pat. No. 5,932,562 by Ostlund, Jr. describes an aqueous micellarmixture of plant sterol and lecithin (in a 1:1 to 1:10 mole ratio) whichhas been dried to a water soluble powder and which is useful as a foodadditive for reducing cholesterol absorption.

U.S. Pat. No. 4,195,084 by Ong describes a taste-stabilizedpharmaceutical suspension of sitosterols to reduce hypercholesterolemia,in which the suspension includes the plant sterol, a chelator such ascalcium disodium EDTA, a surfactant and other ingredients to assuresuspension and dispersal of the phytosterol.

U.S. Pat. No. 3,881,005 by Thakkar et al. describes a pharmaceuticaldispersible powder for oral administration in which sitosterols arecombined with any one of a variety of excipients, and any one of avariety of pharmaceutically acceptable surfactants.

U.S. Pat. No. 6,267,963 by Akashe et al. describes a plantsterol/emulsifier complex that has a lower melting temperature than theplant sterol alone. The complex, e.g., a co-crystallized monoglycerideand plant sterol mixture, is said to facilitate incorporation of thesterol into food products without adversely affecting the texture of thefood products.

As indicated above, it has been widely believed that increasing thesolubility of phytosterols in fat increases their bioavailability andreduces the dose required to achieve a specified degree of cholesterolreduction. Thus, U.S. Pat. No. 5,502,045 by Miettinen et al., describesthe preparation and use of the plant stanol, beta sitostanol, in theform of a fatty acid ester which is readily soluble in an edible oil, toreduce the serum cholesterol level in humans. This technology has beenutilized in manufacturing the margarine product marketed under thetradename Benecol®.

U.S. Pat. Nos. 6,031,118 and 6,106,886 by van Amerongen et al. describesimilar stanol fatty acid esters but provide different and reportedlyimproved chemical methods for their preparation. Plant sterols(fromsoybean oil) have also been interesterified with fatty acid esters toproduce the margarine marketed under the tradename Take Control®.Clinical studies suggest that with mildly hypercholesterolemicindividuals, dietary intake of between 1.5 and 3 grams per day of thefree phytosterol (provided in a fatty acid esterified form) is requiredto decrease plasma cholesterol approximately 15%.

U.S. Pat. No. 5,932,562 by Ostlund, Jr. points out that cholesterol isabsorbed from an intestinal micellar phase containing bile salts andphospholipids which is in equilibrium with an oil phase inside theintestine. Prior to recent experiments, delivery of phytosterol as asolid powder or aqueous suspension was thought to not be preferredbecause of the limited rate and extent of solubility in intestinalliquid phases. In fact, at least two earlier human studies showed thatas much as 9-18 grams of sitosterol per day were required to decreasethe plasma cholesterol level by approximately 15% when the sitosterolwas provided in a coarse powdered (rather than soluble) form. Yet,esterification of phytosterols, coupled with the use of edible oils todeliver these sterols is not always practical, e,g., in formulatingfat-free foods. It is in this context that Ostlund, Jr. provides awater-dispersible mixture of plant sterol and lecithin.

Using a finely milled powdered form of free phytosterols (from tall oil)suspended in a margarine (not fully dissolved or recrystallized in fat),Jones et al. have described cholesterol reduction inhypercholesterolemic humans (Jones et al., Am J Clin Nutr 69:1144-1150,1999) and other mammals (Ntanios et al., Atherosclerosis, 138:101-110,1998; Ntanios et al., Biochim Biophys Acta, 1390:237-244, 1998). Inthese studies, the efficacy based on cholesterol reduction appears to beequal to that of phytosterol and stanol esters reported by others.

Still another method of producing a fine suspension of microparticulatephytosterols in fat and water has been described by Yliruusi et al. inPCT International Publication Number WO 99/43218. The method involvesfirst heating and dissolving beta-sitosterol in a fat or oil, and thenprecipitating the phytosterol with water to form a homogenousmicrocrystalline suspension. While this process appears morecost-effective than grinding, emulsification of fat with water causesany fat to become susceptible to oxidation and necessitatesrefrigeration.

The production of microparticulate phytosterols described in the priorart involves increased cost and inconvenience, e.g., the use ofgrinding, and can result in a mixed emulsified product that is moresusceptible to oxidation and rancidity, particularly when an aqueousfat-phytosterol emulsion is involved. In fact, there are limitations anddisadvantages inherent in most of the above prior methods of phytosterolpreparation and delivery. These methods have included grinding,formation of fat and water mixed phytosterol emulsions, chemicalmodification of phytosterols, e.g., esterification, and mixing ofphytosterols with substantial amounts of specialized solubilizing anddispersing agents.

A recent review article entitled “Therapeutic potential of plant sterolsand stanols” (Plat et al., Current Opinion in Lipidology, 11:571-576,2000) has summarized the results of a number of independent clinicalstudies in which human plasma cholesterol levels were monitored beforeand after ingestion of food products enriched with plant sterols andsterol esters (approximately 2-2.5 g per day). The authors conclude thatLDL cholesterol levels decreased significantly, i.e., an average of10-14%.

The description above is provided to assist the understanding of thereader, and does not constitute an admission that the cited referencesare prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention concerns the use of non-esterified phytosterols infortifying fat-containing prepared foods. Non-esterified phytosterolswere found to have the unexpected property of decreasing the oxidationof fats used in prepared foods, particularly when the fats are heatedand become particularly susceptible to oxidation. It is believed thatsoluble phytosterols e.g., the heat-solubilized non-esterifiedphytosterols described herein, are also able to protect polyunsaturatedfatty acid moieties in fats by quenching, i.e., scavenging, oxidativefree radicals and/or peroxides and hydroperoxides that are formed duringfat oxidation, and that are particularly problematic in heated fats.

Thus, in addition to functioning as a plasma cholesterol-loweringneutraceutical ingredient in prepared foods, phytosterols can actuallyprotect fats against oxidation during cooking and storage. These twodifferent and compatible functionalities each support the novelintroduction of phytosterols into fat-based compositions orfat-containing prepared foods, e.g., into frying and baking shorteningsthat are absorbed (e.g., into potato chips) or otherwise incorporatedinto such prepared foods.

Heat-solubilizing non-esterified phytosterols in fat or oil, followed bycooling and recrystallization, results in formation oftriglyceride-recrystallized non-esterified phytosterols (TRPs). Theinventors have found that when ingested, regardless of the crystallinesize of these fat-recrystallized phytosterols, they were effective atreducing mammalian plasma cholesterol levels. By using cost-effectivenon-esterified phytosterols, and rendering them bioavailable by thermalrecrystallization in fat heating and cooling in the frying fat or in therecipe ingredient fat), the invention provides an effective alternativeto using more costly forms of phytosterols for lowering plasma and livercholesterol levels. Such more costly phytosterols includemicroparticulate powders (ultrafine micron-sized phytosterol powders),chemically modified fat-soluble phytosterols, e.g., fattyacid-esterified phytosterols, emulsified phytosterols, and the moreperishable water-oil microparticulate suspensions of phytosterols.Underlying this new method for utilizing phytosterols is the discoverythat although a chemically unmodified phytosterol (such asbeta-sitosterol) is insoluble in water and poorly soluble in fat, itneed not be converted to a microparticulate powder to be effective atreducing plasma cholesterol levels in vivo.

Accordingly, in a first aspect, this invention provides a prepared foodproduct for ingestion by mammals, e.g., by humans. The food productincludes an oxidation-resistant fat-based composition substantially freeof exogenous solubilizing and dispersing agents for phytosterols. Thisfat-based composition includes between 75% and 98% by weight of at leastone triglyceride-based edible oil or fat, and between 2% and 25% byweight of non-esterified tryglyceride-recrystallized phytosterols(TRPs). At room temperature a limited amount of phytosterol willsolubilize, typically such that a fat will include approximately 1.5% byweight of the phytosterols in solution, with any remaining phytosterolsremaining insoluble. Thus, if phytosterols are added to the fat to alevel from 2% to 25% by weight at room temperature, the fat compositionwill contain approximately 1.5% solubilized phytosterol and between 0.5%and 23.5% by weight of the phytosterols will remain insoluble at thattemperature. Typically the fat-based composition has been partiallyoxidized by an interval of exposure to air during the manufacture andstorage of the prepared food product, and contains a reduced amount ofoxidative by-products compared to a similar fat-based compositionlacking these non-esterified phytosterols.

Storage stability of the food product may also be referred to as theshelf-life of the product at ambient temperatures. Depending upon thefood packaging materials and inert gases utilized in the packagingprocess, the shelf life for such products may range from approximatelyone week to a year or more. This fat-based composition has been shown tobe cholesterol-reducing as measured in the plasma of mammals, and theTRPs when ingested, are essentially as effective, i.e., as bioavailable,as fat-soluble esterified phytosterols in lowering plasma cholesterollevels. Preferably the shelf-life of a prepared food product containingTRPs is increased at least 5%, 10%, 20%, 30%, 50%, 100%, or even morecompared to an otherwise equivalent food product not containing theTRPs.

In particular embodiments, the fat composition includes phytosterols ata level of 2-5%, 5-10%, 10-15%, 15-20%, or 20-25%. In some cases evenhigher levels may be added.

In a related aspect, a prepared food product for ingestion by mammals isprovided as above except that the fat-based or fat-containingcomposition has been partially oxidized by an interval of heating, e.g.,frying, baking, cooking and the like, in air, and contains a reducedamount of oxidative by-products compared to a similar fat-basedcomposition lacking said non-esterified phytosterols. An upper limit forthe interval of heating in air has not been established. However, it isbelieved that any duration of heating of a conventional fat (one that isfree of phytosterols) that results in an acceptable (not excessiveaccumulation of oxidative by-products, (such as free fatty acids andconjugated dienes), will be satisfactory for the phytosterol-fortifiedfat. For example, fats and vegetable oils may be exposed to temperaturesof approximately 180° C. during deep fat frying for periods of timeranging from 5 hr to 25 hr while the prepared food cooked in the oil isexposed to such heat for much shorter intervals, e.g., during cooking(typically several minutes rather than several hours). In any event, aprepared food product as described above may be fried, baked orotherwise heated at least for a time period and to a temperature atleast sufficient to dissolve a desired amount (preferably all) of thenon-esterified phytosterols added to the fat portion of the fatcomposition. The fat composition is substantially free of exogenousphytosterol-solubilizing and dispersing agents. Phytosterol enrichmentof the fat composition decreases the amount of polar and other oxidativeby-products accumulated in the fat and in the prepared food duringheating and exposure to air. At least a portion of the non-esterifiedphytosterols in the fat composition are converted by heating, fullydissolving and subsequent cooling, to triglyceride-recrystallizedphytosterols, i.e. TRPs, in which the TRPs contained in the fatcomposition and in the prepared food product are bioavailable wheningested, to reduce mammalian plasma cholesterol levels.

In certain embodiments, the amount of the edible fat composition in theprepared food product is between 10% and 75% by weight of the foodproduct, e.g., 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, or 60-75%. Inother embodiments, the amount of the edible fat composition in theprepared food is lower or higher, e.g., 1-5%, 2-5%, 3-5%, or 4-5%.

In preferred embodiments, the TRPs are formed by heating at least thefat-based composition (or heating the prepared food product as itcontains the fat-based composition) to a temperature of greater than 60°C., and fully dissolving the non-esterified phytosterols in the fatcomposition, and subsequently cooling this composition to roomtemperature to allow the TRPs to crystallize and be formed.

In another related aspect, a prepared food product for ingestion bymammals is provided that includes a plasma cholesterol-reducing oil orfat composition with improved resistance to oxidation. The oil or fatcomposition is substantially free of exogenous solubilizing anddispersing agents for phytosterols, and includes between 75% and 95% byweight of at least one triglyceride-based edible oil or fat, and atleast 5% by weight of non-esterified triglyceride-recrystallizedphytosterols. As described above, typically the phytosterols are solublein the oil or fat composition at room temperature to a level ofapproximately 1.5% by weight, so that at least 3% by weight ofphytosterols are insoluble at room temperature and have been convertedby heating, fully dissolving, and cooling to formtriglyceride-recrystallized phytosterols, i.e., TRPs. These TRPs, wheningested, are essentially as effective as fat-soluble esterifiedphytosterols in lowering plasma cholesterol levels in mammals.

In preferred embodiments, the oil or fat composition includes at least8%, 10%, 12%, 15%, 17%, or 20% by weight of non-esterified phytosterolsor is in a range defined by taking any two of those values as endpointsof the range. As described above, typically the phytosterols are solublein the fat or oil at room temperature to a level of approximately 1.5%by weight, and the remainder (e.g., at least 6.5%, 8.5%, 10.5%, 12.5%,15.5%, or 17.5% respectively) is insoluble at room temperature, but isdissolved and tryglyceride-recrystallized by heating to dissolve thephytosterols and cooling. These TRPs, when ingested, are essentially aseffective as fat-soluble esterified phytosterols in lowering plasmacholesterol levels in mammals.

In preferred embodiments, the TRPs described above are formed by heatingat least the above referenced oil or fat composition (or a prepared foodproduct containing the oil or fat composition, or the oil or fat and thephytosterols as ingredients of the prepared food) to a temperature ofgreater than 60° C., fully dissolving the non-esterified phytosterols inthe composition, and subsequently cooling the composition to roomtemperature to cause the TRPs to be formed.

In certain embodiments, prepared food products are selected from thegroup consisting of margarine, frying and baking shortenings,mayonnaise, salad dressing, filled dairy products, nut, seed and kernelbutters and chocolate (containing cocoa butter). In each of theseexamples, the phytosterols are dissolved by heating them in the fatportion of these prepared foods, i.e., heating without any aqueouscomponents present. In other embodiments, the prepared food product is apastry or cake.

In certain embodiments, the prepared food product is fried, baked, orotherwise heat-processed with the oil or fat composition, and/or wherethe oil or fat composition and phytosterols are added as ingredients inthe preparation of the prepared food, wherein such heating allows aportion of non-esterified phytosterols that is insoluble in the oil orfat composition at room temperature to be solubilized and thereby enterand be incorporated into the food product, whereupon during cooling,TRPs are formed in the food product.

In preferred embodiments, the prepared food product is selected from thegroup consisting of potato chips, French fries, corn chips, tortillachips, popcorn, and crackers.

Also in preferred embodiments, the food product is cooked, baked, orotherwise heat-processed with the above-described oil or fatcomposition, allowing a portion of non-esterified phytosterols that isinsoluble in the composition at room temperature to be solubilized.During subsequent cooling to room temperature and crystallization ofnon-esterified phytosterols, a partial or complete solidification of theoil or fat composition can occur. This solidification decreases theoiliness, particularly the surface oiliness, perceived by hand contactwith the food product compared to the same food product prepared withoutnon-esterified phytosterols (due to the formation of TRPs in the fat oroil). Solidification or “hardening”of oil can also reduce or prevent oilseparation in certain prepared foods, and is particularly useful in suchfoods as peanut butter, soybean butter, sesame seed butter and otherseed, bean and nut kernel butters. “Hardening” of an edible oil may becompared to that resulting from partial hydrogenation of vegetable oils.Both modifications tend to solidify a vegetable oil by increasing theoil's melting temperature. However, from a nutritional perspective,addition of phytosterols to ones diet advantageously decreases the levelof plasma LDL cholesterol, while addition of partially hydrogenated oilsdisadvantageously increases the LDL level.

In preferred embodiments, the food product, and more particularly theoil or fat composition within the food product, when heated in air, ismore resistant to oxidation and formation of chemically polardegradation products than the same product lacking the non-esterifiedphytosterols, e.g., as described in Example 3 below.

In preferred embodiments, the food product incorporating the oil or fatcomposition has a reduced calorie content compared to a similar foodproduct prepared without non-esterified phytosterols, owing to thepresence of the non-esterified phytosterols that are calorie-free, andsubstitute for a portion of triglyceride-based oil or fat normallyabsorbed or otherwise incorporated into the food product. This statementis explained and supported by Example 4 below.

In preferred embodiments, the non-esterified phytosterols are selectedfrom the group consisting of tall oil-derived phytosterols (such asthose obtained from the manufacture of wood pulp from pine trees) andvegetable oil-derived phytosterols (such as those derived from soybeanoil).

In another aspect, the invention provides an oxidation-resistant fryingor baking shortening that includes: (a) from 75% to 98% by weight of atleast one edible triglyceride-based fat or oil; and (b) from 2.0% to 25%by weight TRPs (produced from at least one non-esterified phytosterolcompound being solubilized by heating, and allowed to recrystallize inthe fat or oil upon cooling). As explained above, typically from 0.5% to23.5% by weight of phytosterols are recrystallized in the solid phase,and approximately 1.5% by weight of non-esterified phytosterol remainssolubilized in the fat at room temperature.

Highly preferably the shortening is substantially free of exogenoussolubilizing and dispersing agents for phytosterols, and the rate offormation of polar oxidation products upon heating the shortening tobetween 160° C. and 190° C. is reduced, compared to the same shorteninglacking the at least one non-esterified phytosterol compound.

Referring to this aspect, the formation of polar oxidation products wasdetermined by measurement of the dielectric constant of the shorteningafter two hours of heating as described elsewhere herein (see Example 3,second experiment). The term “reduced,” referring to the rate offormation of polar oxidation products, indicates that the increase indielectric constant of the shortening is reduced at least 5%, andpreferably 7, 8, or 10% or more for the phytosterol-supplementedshortening, compared to the non-supplemented shortening.

In preferred embodiments, the oxidation-resistant frying or bakingshortening includes at least one edible triglyceride-based fat or oilselected from the group consisting of natural vegetable oils or fats,natural animal fats and oils, structurally rearranged or modifiedvegetable and/or animal fats, and combinations thereof.

In preferred embodiments, the oxidation-resistant frying or bakingshortening includes at least one non-esterified phytosterol compoundselected from the group consisting of vegetable oil-derivedphytosterols, tall oil-derived phytosterols, and combinations thereof.

In preferred embodiments, the oxidation-resistant frying or bakingshortening includes at least one non-esterified phytosterol selectedfrom the group consisting of beta-sitosterol, beta-sitostanol,campesterol, campestanol, stigmasterol, stigmastanol, brassicasterol,brassicastanol, clionasterol and clionastanol, and combinations thereof.

In another aspect, the invention features a method for reducing plasmacholesterol levels in mammals. The method includes providing aheat-processed prepared food containing an edible fat-based compositionthat includes between 75% and 98% by weight of at least onetriglyceride-based edible fat, and between 2% and 25% by weight ofnon-esterified triglyceride-recrystallized phytosterols, for ingestionby the mammal(s). Generally, the phytosterol is soluble to a level ofapproximately 1.5% by weight, such that the insoluble phytosterols inthe fat-based composition at room temperature consititute between 0.5%and 23.5%. The fat-based composition is substantially free of exogenousphytosterol-solubilizing and dispersing agents. The insolublephytosterols have been heat-solubilized and subsequently cooled to formtriglyceride-recrystallized phytosterols i.e., TRPs. The TRPs wheningested are essentially as effective as fat-soluble esterifiedphytosterols in reducing plasma cholesterol levels.

In preferred embodiments, the proportion of non-esterified phytosterolsused in the edible fat-based composition for a prepared food is between3% and 15% by weight of the composition, and more preferably between 5and 10% of the composition (or other percentage as described for foodproducts herein). Thus, with the latter range, a serving of foodcontaining 10 grams of a fat-based composition, would contain between0.5 g and 1.0 g of non-esterified phytosterols. This amount isconsistent with current recommendations published by the U.S. Food andDrug Administration.

The edible fat-based composition is heated to a temperature of greaterthan 60° C., and preferably between 75° C. and 150° C., or higher, todissolve the non-esterified phytosterols in the composition. At atemperature of 60° C. or below, the rate of dissolution is slower thandesirable, and the concentration of dissolved phytosterols in afat-based medium is lower than generally desired to be commerciallyuseful or practical.

In preferred embodiments, between 0.5 g and 4.0 g of the non-esterifiedphytosterols contained in the above prepared food are ingested daily byhumans.

In preferred embodiments, the TRPs are formed by heating at least theedible fat-based composition to a temperature exceeding 60° C. for aperiod of time sufficient to dissolve the non-esterified phytosterols inthe fat, and subsequently cooling the composition (or the foodcontaining this composition) to room temperature to cause the TRPs to beformed.

In a related aspect, the invention features a method for reducing plasmacholesterol levels in mammals, including providing and regularlyingesting a heat-processed prepared food containing an edible fat-basedcomposition that contains between 75% and 97% by weight of at least onetriglyceride-based edible fat, and at least 3% by weight ofnon-esterified triglyceride-recrystallized phytosterols. Typically thephytosterols are soluble in the fat at a level of approximately 1.5% andthe remainder (e.g., 1.5% from a total of 3%) is insoluble at roomtemperature. The fat-based composition is substantially free ofexogenous phytosterol-solubilizing and dispersing agents. The insolublephytosterols are heat-solubilized and subsequently cooled to formtriglyceride-recrystallized phytosterols, i.e., TRPs. The TRPs wheningested are essentially as effective as fat-soluble esterifiedphytosterols in reducing plasma cholesterol levels.

In certain embodiments, the fat composition contains at least 5%, 7%,10%, 12%, 15%, 17% or 20% by weight of non-esterified phytosterols(typically the phytosterols are soluble to a level of approximately 1.5%at room temperature and the remainder is insoluble).

In another aspect, a method is provided for preparing a TRP-containingfat-based composition. The method includes (i) providing atriglyceride-based edible fat-containing composition that includesbetween 2% and 25% by weight of non-esterified phytosterols and not morethan 98% by weight of edible fat or oil, in which the composition issubstantially free of exogenous phytosterol-solubilizing and dispersingagents, (ii) heating the composition to dissolve (preferably fullydissolve) the non-esterified phytosterols, and, (iii) cooling thecomposition to room temperature, allowing formation of TRPs. Typicallythe phytosterols are soluble in the edible fat or oil at roomtemperature to a level of approximately 1.5%, while the remainder isinsoluble at room temperature. In general the fat-containing compositionis heated to a temperature of 60-180° C., usually 75-150° C.

Similarly, in a related aspect the invention concerns a method ofpreparing a TRP-containing fat-based composition by heating a mixture ofa triglyceride-based edible fat-containing composition, andnon-esterified phytosterols, where the mixture includes not more than98% by weight of edible fat or oil and 2% to 25% by weight ofnon-esterified phytosterols for sufficient time and temperature todissolve said non-esterified phytosterols, and cooling the compositionto room temperature.

In yet another aspect, a method is provided for preparing anon-esterified phytosterol-fortified prepared food. The method includes:(i) providing an edible fat-based composition that includes between 2%and 25% by weight of non-esterified phytosterols and between 75% and 98%by weight of at least one edible fat or oil, in which the composition issubstantially free of exogenous phytosterol-solubilizing and dispersingagents, and one or more other ingredients for the prepared food if anysuch additional ingredients are used; (ii) cooking or otherwise heatingthe prepared food ingredients with the composition to allow thenon-esterified phytosterols to dissolve in the oil or fat and enter orbecome integrated into the food product; and (iii) cooling the foodproduct to room temperature to allow formation of TRPs in thecomposition within the prepared food.

In certain embodiments, the fat-based composition can be used as aningredient mixed with other ingredients in the preparation of theprepared food, and/or the prepared food product can be cooked in thefat-based composition.

While in most cases the non-esterified phytosterols are recrystallizedin the oil or fat prior to combining with other ingredients, for someprepared foods, the phytosterols can be combined with the oil or fat inpreparation of the prepared food. Thus, alternatively, the fat or oiland the phytosterols can be added as separate ingredients in such mannerthat the phytosterols will dissolve in the fat or oil upon heating ofthe combined ingredients. In some cases, only a portion of thephytosterols added as ingredients will become solubilized, where only aportion of the phytosterols are in contact with the fat or oil duringheating. In cases where the fat-based composition, or the oil or fat andthe phytosterols are added as ingredients in preparing the preparedfood, typically a number of different ingredients are blended or mixedsuch that the various ingredients are relatively uniformly distributedthroughout the mixture.

Another aspect concerns a method of increasing the oxidative stabilityof a heated frying fat composition, where the method involvesmaintaining a fat composition that contains at least 8% by weightnon-esterified phytosterols at a temperature of at least 100 degrees C.,wherein said fat composition is used for frying.

The frying fat composition can be held at the elevated temperature for asuitable length of time considering the purpose, e.g., at least 0.5 hr,1 hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs, or longer. Of course, as withany frying fat composition, eventually the fat will degrade sufficientlythat it will not be used any longer for frying, and may he replaced withfresh fat composition. In particular embodiments, the fat compositionoxidizes at a rate that is only 90, 80, 70, 60, 50, 40, 30, 20% or evenless of the rate for the same fat composition without phytosterols orother non-fat oxidation rate reducing components.

In the particular embodiments, the fat composition containingnon-esterified phytosterols is a composition as described for otheraspects herein.

Yet another aspect concerns a dietary supplement that includes at leastone triglyceride-based edible fat, and between 3% and 50% by weight oftriglyceride recrystallized phytosterols. Such a dietary supplement canalso be regarded as a nutraceutical. The supplement can be in numerousdifferent forms, e.g., capsule, pill, wafer. The TRP-fat composition canbe combined with other dietary components, such as protein, vitamins,minerals, and combinations of such components.

In certain embodiments, the phytosterol content, fat content,preparation method for the composition, and other parameters are asdescribed herein for other aspects involving a fat/TRP composition.

The term “prepared” in the context of a “prepared food product” refersto a commercially processed and packaged food product containingmultiple combined ingredients, in which the processing includes at leastone step in which the assembled food product (or one or moretriglyceride-based fat or oil ingredients that are either contacting, orbeing combined into the food product), are heated together with asuitable quantity of phytosterol ingredient(s), to a temperaturesufficient to dissolve the phytosterols in the fat or oil, and oftensubstantially higher than this temperature, and for a period of timesufficient to process, cook, fry or otherwise complete theheat-preparation of the food product. Upon cooling, a portion of thephytosterols recrystallize in a fat or oil component of the processedprepared food product. Examples of such prepared food products includepotato chips (containing at least potatoes, frying fat or oil, andphytosterols), French fries, corn chips, tortilla chips, popcorn,crackers, peanut butter, soybean butter, sesame seed butter and othernut kernel butters, mayonnaise, processed cheese, chocolate and thelike.

The term “fat” may be used broadly and generally, referring to an edibletriglyceride that may be either liquid (also specifically termed oil) orsolid at room temperature (also specifically termed fat), and that isderived from a single vegetable source (e.g., soybean, cottonseed, corn)or an animal source (beef tallow, pork lard) or a blended combination ofsources. Unless specifically limited to edible triglyceride compositionsthat are solid at room temperature, use of the term “fat” includes oils.Also unless clearly indicated to the contrary, the term “fat” alsoincludes chemically and enzymatically modified triglyceride-based liquidand solid fats and blends thereof (e.g., hydrogenated, partiallyhydrogenated, chemically or enzymatically interesterified, or assembled,i.e., “structured” triglycerides and combinations thereof.

The phrase “improved resistance to oxidation” for a fat that containsnon-esterified phytosterols refers to a fat exhibiting at least a 10%reduced rate of degradation by oxidation in air, compared to oxidationof the same fat without phytosterols. This differential oxidation rateis particularly evident during heating of the oil, e.g., frying with theoil at a temperature of 160-190° C. Oxidation rate is evidenced by oneor more physical measurements such as dielectric constant measurement ofpolar oxidation products formed in the fat, AOM (accelerated oxidationmeasurement, OSI (oxidative stability index), or organoleptic quality(tasting for rancidity). The extent of oxidative protection provided bynon-esterified phytosterols dissolved in fat heated to 180° C. is afunction of the type of fat and the concentration of phytosterols in thefat. Improved resistance to oxidation is particularly evident in avegetable oil containing polyunsaturated fatty acids, e,g., soybean,corn and canola oil. When 10% by weight soybean-derived phytosterols isdissolved in such oils, the rate of oxidation, i.e., formation of polaroxidation products, in the heated oils is at least 10% lower than therate in the same oil lacking phytosterols. Preferably, the rate ofoxidation is at least 20% lower, and more preferably, the rate is 30%,40% or even 50% lower than the rate in the same oil lackingphytosterols.

The term “partially oxidized” refers to a fat-based composition that hasbeen exposed to air either with or without heating, e.g., frying orbaking and that has at least begun to accumulate oxidative by-productswhose concentrations are measurable either in the oil or in the vaporabove the oil by conventional means, e.g., by conductivity, dielectricconstant, and free fatty acid content.

It is believed that oxidative protection of fats and oils provided byphytosterols has not been reported previously. Also, phytosterols arenot recognized as antioxidants or as scavengers or quenchers offree-radicals or peroxides and hydroperoxides formed during oxidation ofpolyunsaturated fatty acid moieties. In searching for a rationalexplanation for this oxidative protection, Applicants have looked toliterature describing various properties of cholesterol. Of course“cholesterol fortification” of a food product would be nutritionallyundesirable and, indeed, phytosterol fortification is intended to reducecholesterol uptake. However, the cholesterol molecule is structurallyrelated to the phytosterols, i.e., addition of an ethyl side group tobeta-sitosterol generates cholesterol. U.S. Pat. No. 6,214,534 byHorowitz et al. describes several UV light photodynamic quenchersincluding vitamins, thiols, cholesterol, and several other compoundsthat react with, and inactivate both free radicals and reactive forms ofoxygen. Since free radicals, peroxides and hydroperoxides are producedduring the oxidation of polyunsaturated fatty acid groups intriglycerides, phytosterols dissolved in fat may inactivate thesereactive compounds, as with cholesterol described in the photodynamicsystem of Horowitz et al. While the phytosterols may act in this manner,the present invention is not limited by this explanation.

The term “edible” in the context of an oil or fat-based compositionmeans that the composition is suitable for use in mammalian, e.g.,human, foods, dietary supplements and pharmaceutical preparations.

The term “exogenous phytosterol-solubilizing and dispersing agents”refers to agents other than triglycerides in the prior art, that havebeen added to triglyceride-based oils and fats to promote thecholesterol-lowering efficacy of phytosterols (see discussion above inthe Background section). A partial list of these agents includesmonoglycerides, diglycerides, lecithin, vitamin E, the sorbitans andother surfactants, and fatty acids chemically esterified withphytosterols.

The term “substantially free,” referring to any presence of exogenoussolubilizing and dispersing agents for phytosterols, means that eitherzero percent, or in any event, less than 50% (and preferably less than25%) of the amount of such an agent or agents that would be required inthe absence of triglycerides, to achieve solubilization or dispersal ofnon-esterified phytosterols (at room temperature) that have been addedto the referenced composition. Provided that the phytosterols arerecrystallized in triglycerides, triglycerides alone are sufficient forphytosterol bioavailability, i.e., effectiveness in plasma cholesterolreduction. Therefore, any addition of such a non-triglyceridesolubilizing or dispersing agent to a fat-based composition containingTRPs is considered gratuitous and optional.

The term “phytosterol” refers to any of a group of sterols derived fromplants (see examples below in Example 1).

The term “non-esterified phytosterols” refers to forms of phytosterolsthat are free of ester chemical side chains. Conversely, esterifiedphytosterols are most commonly fatty acid-esterified phytosterolsmanufactured to promote phytosterol solubility in fat. Non-esterifiedphytosterols are defined herein to include both the non-esterifiedsterol and stanol forms of phytosterols (see Example 1 below). Accordingto the present invention, phytosterols are dissolved in oil or fatbefore recrystallization, and therefore the particle size, texture, etc.of the material can be coarse for reasons of economy, i.e., chemicaldissolution reduces the material to molecular dimensions. Dissolution ofmore costly forms of phytosterols, e.g., ultrafine micron-sizedphytosterol powders, would be economically wasteful, but can also bedone.

The composition which includes between 75% and 98% by weight of at leastone triglyceride-based edible oil or fat, allows between 2% and 25% byweight of non-esterified phytosterols to be added to the samecomposition. A 3% to 10% by weight concentration range is a preferredrange. Accordingly, at the 3% level, a food that contains 1 Og of fatper serving will provide at least 0.3 g of phytosterols per serving. Inthe case of pharmaceutical preparations, the composition may include aslittle as 50% by weight of at least one triglyceride-based edible oil orfat, to allow between 3% and 50% by weight of non-esterifiedphytosterols to be added to the same composition.

The process of treating the non-esterified phytosterols by “heating,fully dissolving, and cooling” refers to a process that: (i) heats thephytosterols together with triglyceride-based fat or oil (and optionallyother food ingredients constituting a prepared food product) to atemperature of greater than 60° C. until the phytosterols havedissolved, and then (ii) cooling the heated product and allowing thetriglycerides to associate with the recrystallizing phytosterols.Flash-chilling with chilled air or with a chilled water jacket may tendto precipitate and segregate the phytosterols from the triglycerides,preventing optimal recrystallization. Conventional or normal ambient aircooling rates of prepared foods containing heated triglycerides andphytosterols is preferable to flash cooling. For example,in may casescooling of a fat-based composition or prepared food to room temperaturewill occur over a period of 5 minutes to 2 hrs, although longer orshorter times can be used.

The term “triglyceride-recrystallized phytosterols” or TRPs and theprocess of heating and cooling these ingredients is described elsewhereherein. The term “recrystallized” is distinguished from the term“solubilized”(in which the phytosterols are dissolved to form a clearsolution). Recrystallized is meant to indicate that the phytosterolsafter initially being dissolved in one or more triglyceride-based fatsor oils, are allowed to cool and recrystallize in the oil or fat. Byphysical analyses (light microscopy of lipid stained crystals, andmelting temperature determinations described elsewhere herein),Applicants have determined that such recrystallization results in fatsand/or oils, i.e., triglycerides, becoming intimately associated withcrystallizing phytosterols. The resulting products are mixed and/orinterrupted crystal structures having melting temperatures reduced belowthat of the phytosterols alone. It is believed that these physicallydestabilized, triglyceride-containing crystals are more easilyemulsified and/or dissolved in the mammalian gut, resulting in improvedphytosterol bioavailability and therefore more effective plasmacholesterol reduction in vivo. As noted above, a proportion of thephytosterols is soluble in the fat at room temperature (typically at aconcentration of about 1.5%). Therefore, when a combination ofphytosterols and fat is heated to dissolve solidified (crystallized)phystosterols and then cooled, phytosterols that cannot remain insolution at room temperature solidify or recrystallize, but a portionremains dissolved in the fat. Thus, unless clearly indicated to thecontrary, reference herein to “triglyceride-recrystallized phytosterols”or “TRPs” includes both the dissolved phytosterols as well as there-solidified or recrystallized phytosterols.

The term “effective” refers to the extent to which plasma cholesterollevels in mammals are reduced by regular, e.g., daily, twice daily, orthrice daily ingestion of the recommended 1-2 gram dose (or theappropriate divided dose) of phytosterols. In a random population ofhuman adults, a 5% to 15% or greater lowering of total cholesterol inthe plasma caused by ingestion of phytosterols is considered effective.

The term “esterified phytosterols” refers to phytosterols (plant sterolsand stanols) that have been joined through an ester linkage to fattyacids using a chemical, enzymatic, combination, or other process. Thecommercial margarines Benecol® and Take Control® discussed above,incorporate such esterified phytosterols. Therefore, “non-esterifiedphytosterols” refers to phytosterols that have not been esterified tofatty acids as described.

The term “reduced surface oiliness” means that upon routine handling ofthe prepared food, less oil is transferred from the food to ones hands(or to an absorbant surface) than would otherwise occur if the food wereprepared with the oil or fat alone (see Example 5 below).

As used herein, the term “dietary supplement” refers to a preparationthat is adapted to supplement an individual's diet by providing one ormore dietary components. A “nutraceutical” refers to a product isolatedor purified from foods, and generally sold in medicinal forms notusually associated with food and demonstrated to have a physiologicalbenefit or provide protection against chronic disease. In the presentinvention, phytosterols provide a hypocholesterolemic benefit and are anutraceutical.

For the definition of any other fat and oil-related terms is that havenot been defined herein, the reader is referred to the reference book,Bailey's Industrial Oil and Fat Products, Fourth Edition, Daniel Swern,editor, John Wiley & Sons, N.Y., 1979.

By “comprising” is meant including, but not limited to, whatever followsthe word “comprising”. Thus, use of the term “comprising” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of”.Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements may be present. By“consisting essentially of” is meant including any elements listed afterthe phrase, and limited to other elements that do not interfere with orcontribute to the activity or action specified in the disclosure for thelisted elements. Thus, the phrase “consisting essentially of” indicatesthat the listed elements are required or mandatory, but that otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Additional aspects and embodiments will be apparent from the followingDetailed Description and from the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Recently, a number of investigators have described a variety of methodsfor producing very small particles or microcrystals of phytosterols. Itis believed that such small particles have greater efficacy in beingdispersed in the GI tract and controlling plasma cholesterol levels.U.S. Pat. No. 6,129,944 by Tiainen et al. describes the production of amicrocrystalline phytosterol product useful as a cholesterol-loweringagent, formed by pulverizing, i.e., dry or wet grinding, a crystallinephytosterol to produce microparticles having a preferred mean particlesize of approximately 5-10 microns. The microcrystalline phytosterolproduct can be mixed with a sweetening agent and water or alternatively,mixed with another carrier such as fat to form a microparticulateemulsion. There is no suggestion by Tiainen et al. or any otherinvestigator of which the inventors arc aware that microcrystallinephytosterols after being formed, should be heated or dissolved in such afat or oil. Such heating in oil, as described for the present invention,would be expected to destroy the sized microparticles described byTiainen et al.

As described herein, phytosterols are recrystallized with triglyceridesvegetable oil, shortening, or the like). The first step involves heatingthe triglyceride(s) and phytosterol(s) until the phytosterols aredissolved. This phytosterol-triglyceride solution is used to contact, orbe combined with the food product being fried, cooked or otherwiseheated. (Alternatively, the fats and the phytosterols are added asseparate ingredients in the preparation of a prepared food.)Subsequently, the prepared food product is cooled (preferably bycontacting the heated food product with ambient air). Under the lightmicroscope (600× magnification), it is seen that phytosterols that havebeen recrystallized in vegetable oil, e.g., soybean oil, tend to form adiversity of macrocrystalline structures spanning tens or hundreds ofmicrons. This material when tasted, has a surprisingly soft andagreeable mouth feel, and includes elongated hexagonal crystals,radially extending branched crystalline needle structures (appearing aswispy ball-shaped structures), and large extended flat plate crystals.On the other hand, phytosterols that are recrystallized byquick-chilling to room temperature (e.g., by ice chilling to roomtemperature in a few seconds rather than by ambient air contact), tendto form harder, smaller, more homogeneous needle-like micro-crystalshaving diameters of only a few microns, i.e., 1-4 microns.

The temperature required to re-dissolve the above crystals in thesurrounding vegetable oil differs significantly depending upon therapidity of recrystallization. For example, 10% by weightsoybean-derived phytosterols that were recrystallized at roomtemperature in soybean oil, redissolved in the oil at a temperature of65° C. On the other hand, the more rapidly ice-recrystallizedphytosterols described above required a higher temperature (72° C.) tobe redissolved. By comparison, the same amount of phytosterol (as a drypowder) initially placed in soybean oil, required a temperature ofnearly 85° C. to be dissolved. The observations on recrystallization(coupled with the microscopic analysis of crystalline sizes and shapes)suggested that slower recrystallization allows formation of mixedcomposition triglyceride-containing (larger) phytosterol crystals. Thesecrystals would be expected to redissolve more easily, i.e., at a lowertemperature, than the rapidly formed crystals.

To determine whether the larger crystals contained any triglycerides,these crystals were washed and centrifuged twice in ethanol. Next, thecrystals were stained with a saturated Sudan Black solution (60% byweight ethanol in water) to visualize any lipids. Light microscopyconfirmed that the lower melting point larger crystals (but not thehigher melting point small needle-shaped crystals) contained multipleinternal layers and occlusions of lipid. It is reasonable to concludethat the intimate association of triglycerides and phytosterols thatresults from fully dissolving and then recrystallizing phytosterols infats, yields crystals having a reduced melting temperature. Thesecrystals appear to provide dietary phytosterols in a highly bioavailableform useful for reducing plasma cholesterol levels.

While it has been recently reported that a crystalline complex can beformed by combining phytosterols and monoglyceride emulsifiers (seeabove, U.S. Pat. No. 6,267,963), the existence and utility oftriglyceride-recrystallized phytosterols have not been previouslydescribed. In fact, Applicants have not found any prior reference toformation of a mixed crystalline complex or association betweentriglycerides and phytosterols that enhances phytosterolbioavailability.

Non-esterified phytosterols are known to have a very limited solubility(to a concentration of approximately 1.5% by weight) in an edible oil orfat at room temperature. Nevertheless, between 2% and 25% by weight ofnon-esterified phytosterols (e.g., semi-pure or purified phytosterolsfrom soybeans or pine tree tall oils), can be readily and convenientlydissolved in edible oil or fat by heating to a temperature of 60° C. orgreater, and preferably 75° C.-100° C. or above (the requiredtemperature depending upon the concentration of phytosterols to bedissolved). Subsequently, as the heated composition is cooled to roomtemperature, a substantial portion of the solubilized phytosterolprecipitates, i.e., is recrystallized, in the triglyceride-based oil orfat in the form of a Triglyceride-Recrystallized Phytosterol compositionor complex (hereinafter abbreviated “TRP”, “TRP composition or TRPcomplex”).

Remarkably, the TRP composition formed in this manner has been found tobe as potent in the mammalian diet at reducing the levels of plasma andliver cholesterol as fatty acid-esterified phytosterols that are fullysoluble at room temperature. In the first direct comparison betweennon-esterified phytosterols and equivalent amounts of phytosterols assterol esters in the same experiment, it was found that non-esterifiedphytosterols fully dissolved in oil by heating (>60 degrees C., andpreferably >80 degrees C.), and provided equivalent (or even greater)reductions in plasma and liver cholesterol as compared to equivalentamounts of esterified sterols. In the context of cholesterol reduction,the term “greater” means that the cholesterol reductions measured andreported herein and in the Hayes reference are greater than thosereported by Ntanios and Jones (Biochim. Biophys. Acta (1998)1390:237-244) for the same levels of sterols, in which the sterols wereincompletely dissolved in fat. While TRPs may have been accidentallyproduced in the past in the course of heating and cooling non-esterifiedphytosterols and fats, their utility for plasma cholesterol reductionwould not have been recognized due to their poor room temperaturesolubility.

The presently described TRP composition is more convenient andcost-effective than esterified phytosterols or phytosterol-containingcompositions that have been supplemented with solubilizers, emulsifiers,antioxidants and other additives for inclusion in foods. The TRPcomposition also has a significant advantage over the finely milled andmicrocrystalline powdered forms of phytosterols described by Tiainen etal. and Jones et al., in light of the considerable cost associated withproducing these micron-sized powders. The present composition isparticularly useful in preparing fat-based foods such as shortening,margarine, mayonnaise, salad dressing, peanut butter and the like, andprocessed food products including fried and baked snack foods.

Surprisingly, as illustrated below, the presence of dissolvedphytosterols in a heated oil or fat, improves the triglyceride'soxidative stability, and at ambient temperature, decreases the surfaceoiliness of foods fried in the triglyceride-based composition. At thesame time, the caloric fat content of a food prepared in or with theTRP-containing composition is reduced. While other investigators havefound that finely milled or microcrystalline preparations ofnon-esterified phytosterols that have not been initiallyheat-solubilized in an oil or fat, can also function efficiently toreduce mammalian plasma cholesterol levels, the additional benefitsdescribed above are obtained only after heat-solubilization. Forexample, heat-solubilization in a triglyceride-based edible oil allowsnon-esterified phytosterols to freely enter a food product as it isbeing fried in the oil, whereas particles of phytosterols would beexcluded. Likewise, suspended particles would not be expected to improvethe oxidative stability of an oil.

For the purpose of this invention, the fat or oil used as a vehicle orcarrier for the phytosterol herein, is a conventional triglyceride-basedcooking fat or oil that is substantially free of phytosterolsolubilizing agents, dispersants and/or detergents (collectively termed“oil emulsifiers or additives”). Examples of such fats and oils includenatural vegetable oils, interesterified fats and oils, and partiallyhydrogenated vegetable oils, animal fats and combinations thereof.

Unlike recently described compositions for oils and fats containingphytosterols described above in the Background, the presently describedtriglyceride-based composition contains substantial amounts of insolublephytosterol (recrystallized in fat) rather than solubilized phytosterol,and is substantially free of the above-described oil additives fordispersing or solubilizing phytosterols. The composition is particularlyuseful in preparing fat-containing foods that do not require oiltransparency at ambient temperatures. This is true of margarines,shortenings, mayonnaise, cheese and other dairy fat-containing products,some salad dressings, and many other foods including processed foodsthat are fried, baked or otherwise prepared by cooking or heating in, orin combination with fat or oil. Examples of such foods include the snackfood category, e.g., potato chips, crackers, and the bakery category,e.g., donuts, pies, cakes, and the like.

The present invention describes compositions and methods for introducingsubstantially fat-insoluble non-esterified phytosterols into foods,including snack foods, by means of the standard fat or oil that is usedin the frying or baking of such foods. It was the inventors' intentionto compare the efficacy of using non-esterified phytosterol preparationsrecrystallized in edible fat and used in foods, e.g., fried foods, withthat of more costly diglyceride-solubilized or fatty acid esterifiedphytosterols in limiting cholesterol absorption in the gut, and loweringplasma cholesterol levels. Surprisingly, the phytosterols recrystallizedin fat that has been incorporated into such foods are very effective,i.e., bioavailable, in reducing plasma and liver cholesterol levels. Itis believed that this cholesterol-lowering efficacy compares favorablywith that of fully solubilized phytosterol preparations (e.g.,phytosterols esterified with fatty acids to assure solubility infat-containing products such as Benccol® and Take Control® margarines).

As an unanticipated benefit and utility in the present invention, thepresence of 5-10% or more by weight of phytosterol that has beenrecrystallized with triglycerides in the oil portion of fried snack food(e.g., potato chips) has been found to decrease the surface oiliness offried food when compared to food fried in oil lacking the phytosterol.Applicants have also found that the presence of either soybeanoil-derived phytosterols or tall oil-derived phytosterols in vegetableoil during frying, helps in chemically stabilizing the oil againstoxidation by reducing the rate of appearance and the amount of polarbreakdown products in the oil. To the extent that the phytosterolsreplace a portion of the oil in such a blend, the phytosterols alsoserve to reduce the caloric fat content of a food cooked in the blend.Thus, the present invention also provides methods for decreasing thesurface oiliness of fried foods, and the resulting fried foods, andmethods for providing reduced calorie food, utilizing TRPs as describedherein.

Except for micron-sized finely milled powders of non-esterifiedphytosterols described by Tiainen et al. and Jones et al. (see above),as well as previously described emulsified preparations, thenon-esterified phytosterols have been thought to lack “bioavailability”relative to esterified sterols and stanols, as emphasized in theintroductory references. In this instance, bioavailability for a givenquantity of phytosterol means the potency of that particular physicaland/or chemical form of phytosterol in lowering the plasma level oftotal and LDL cholesterol. Despite the limited solubility ofnon-esterified phytosterols in fats and oils at room temperature, it hasbeen discovered that concentrations of between 2% and 25% by weightnon-esterified phytosterols (e.g., soybean oil-derived mixed prilledsterols or stanols or tall oil-derived sterols and stanols) can beconveniently and rapidly dissolved by mixing or other agitation indiverse oils, fats and fat-containing foods, e.g., cooking or salad oil,shortening, peanut butter and dairy cream, heated to a temperature ofgreater than 60° C., and preferably between 75° C. and 150° C., orabove. At higher temperatures such as 180° C., a heated oil or fat,e.g., corn, canola, cottonseed, soybean oil, or palm oil that containsheat-solubilized phytosterols is useful in the preparation (e.g., fryingand baking) of potato chips and other snack foods. When suchheat-solubilized phytosterols are cooled and recrystallized in such fatsor fat-containing foods, their ability to lower plasma cholesterollevels is excellent (see nutritional studies below).

The fat compositions and food products of the present invention can beprepared by conventional methods, with the addition of phytosterols(e.g., as described herein). Persons familiar with preparation of fatcompositions and food products can routinely select suitable componentsfor a particular product.

Preliminary Study, Reducing Plasma Cholesterol Using Non-EsterifiedPhytosterols and an Emulsifier in Dietary Fat.

The efficacy of adding 0.25% by weight soybean oil-derived prilledsterols and 0.25% soybean prilled stanols to a hamster diet containing0.05% cholesterol to reduce the animal's plasma cholesterol level wasinvestigated. Hamsters were fed a cholesterol-containing diet in whichthe dietary fat (30% soybean oil, 50% palm oil and 20% canolaoil-providing approximately equal amounts of saturated, monounsaturatedand polyunsaturated fatty acids) was either supplemented orunsupplemented with up to 6% by weight of an emulsifying agent(subsequently reported by Goto et al. in U.S. Pat. No. 6,139,897) toenhance the solubilization of sterols and stanols in the fat portion ofthe diet. It was expected that this agent, a mono- and diglycerideemulsifier (40% glyceryl monocleate+60% glyceryl dioleate), whichreadily dissolves both sterols and stanols, would enhance the ability ofthese phytosterols to lower hamster plasma cholesterol levels.

Surprisingly, each cholesterol-lowering regimen (i.e., sterols andstanols, each tested separately after heating with dietary fat; orstanols combined with either 3% or 6% by weight of the above emulsifierin the heated dietary fat) was found to reduce the plasma cholesterollevel to the same extent. More specifically, while the plasma totalcholesterol value (TC) in hamsters fed a cholesterol-supplemented dietwas found to average 185 mg/dL, and the TC value in hamsters fed acholesterol-free diet averaged 135 mg/dL, all of the dietary regimensincorporating a low level (0.25% by weight) of phytosterols (5:1sterol-to-cholesterol) resulted in significantly reduced TC valuesaveraging 160±15 mg/dL. (Liver E C, i.e., esterified cholesterol, showedthat 1:3 monoglycerides improved efficacy, as well) These resultssuggested that phytosterols can function effectively to lower TC bothwhen they are solublized in the diet (e.g., using mono- and diglyceridesadded to a dietary fat) and when they are recrystallized in thetriglyceride (fat) portion of the diet, after being initiallysolubilized in the heated fat. It is also possible that finely milledmicron-sized powder phytosterol preparations would function well tolower TC (without fat recrystallization), but these preparations havethe disadvantage of greater manufacturing cost.

Examples Example 1 Phytosterol Preparations and Solubilities in CookingOil

Two industrial samples of non-esterified phytosterols were used in aseries of experiments described below. These samples included soybeanoil-derived mixed prilled phytosterols and mixed prilled stanols (thelatter prepared by fully hydrogenating the former). Both were obtainedfrom ACH Food and Nutrition, Inc., Memphis, Tenn. The soybeanoil-derived prilled phytosterols containing up to 4% by weightbrassicasterol, 30% campasterol, 20% stiginasterol, and 40%beta-sitosterol.

The limit solubility of each phytosterol in cooking oil was measured byfully dissolving a graded series of concentrations (from 1% to 5% byweight, in steps of 0.5%) of each sample in soybean oil heated to 150°C., then cooling the samples to room temperature and waiting 24 hoursfor any supersaturating phytosterol to crystallize. All phytosterolsappeared soluble in room temperature cooking oil at a concentration of1.5% by weight, while all showed precipitates at concentrations of 2.0%and higher.

It is generally appreciated that at least 1-1.5 grams per day ofphytosterol must be consumed by humans to achieve a useful decrease,e.g., a 5%-15% decrease, in the plasma cholesterol level. If one is toobtain this phytosterol dose in, for example, two 1 ounce servings of afood product rich in fat, e.g., a snack food containing 30% by weightfat, then the fat should contain approximately 7% by weight (or more)phytosterols (7% phytosterol×30% fat×56g food=1.2g phytosterol). With alimit solubility of approximately 1.5% in room temperature oil, most ofthis 7% level of phytosterol crystallizes in a conventional cooking oilor fat as it cools.

In the hamster, rabbit and human nutritional studies by Jones et al. andNtanios et al. (cited above), non-esterified phytosterols provided indietary fats caused a significant reduction in plasma cholesterollevels. Whether these phytosterols were simply suspended in the dietaryfat as indicated in the rabbit and human studies, or alternatively,dissolved as suggested in the hamster study, (and described in thepresent invention), was investigated. Applicants prepared thecoconut-olive-sunflower fat blend specified by Ntanios et al. in theirhamster study, mixed it with the specified amount of tall oil-derivedphytosterols (1 part by weight phytosterol and 5 parts by weight of thefat blend), and heated the resulting 17% by weight phytosterolsuspension to 60° C., also as specified. After 4 hours heating, thesuspension appeared unchanged, i.e., undissolved. It has been concludedthat the bulk of phytosterols used by Ntanios et al. were suspended inoil rather than being dissolved and recrystallized.

In fact, Applicants have determined that little more than 2-3% by weightphytosterols can be conveniently dissolved in fat when heated only to60° C. Temperatures greater than 60° C. are suggested for fullydissolving these higher concentrations of phytosterols in fats and oils,and preferably temperatures of 75° C., 100° C. or even greater to speedthe solubilization process prior to allowing recrystallization to occur.Within the scope of the present invention, for much more dilutephytosterol suspensions than those described by Ntanios et al., i.e.,for 2%-6% by weight phytosterol suspensions in fats and oils,temperatures as low as 50° C.-60° C. may eventually promote phytosterolsolubilization, albeit at a much slower rate than solubilization at 75°C.-100° C., prior to cooling and formation of TRPs.

Example 2 Crystalline Phytosterol Composition Formed with Triglycerides

One part by weight tall oil-derived phytosterol or one part by weightsoybean-derived prilled phytosterol powder (non-esterified phytosterols)described above were each heated with nine parts soybean oil. Thetemperature required to solubilize these 10% by weight powders in oilwas approximately 75-85° C. From Example 1 it was estimated thatapproximately 8.5% by weight phytosterols (out of 10% total)recrystallized in the oil following cooling to room temperature. Phasecontrast microscopic examination (600× magnification) of the solidsshowed a mixture of extended needle and plate-type crystalline materialsuspended throughout the mixture, that differed markedly from theamorphous solids originally placed in the triglyceride oil.

Upon reheating, much of the precipitated crystalline material appearedto redissolve very quickly at a temperature 10-20° C. below the originalsolubilization temperature for the phytosterol powders. Thus,phytosterols first heated and dissolved, and then recrystallized intriglyceride oils appear to be more readily heat-dispersible thanpurified phytosterol powders. This observation supports the hypothesisthat a crystalline phytosterol composition is formed in (or togetherwith) triglycerides, that may be more bioavailable and effective in themammalian gastrointestinal system than phytosterol alone for reducingcholesterol absorption.

The limited bioavailability of non-solubilized phytosterol powder isevident in the earlier research of Faquhar et al., Kucchodkar et al.,and Lees et al. (cited above in the Background). Their researchindicated that nine or more grams of phytosterol powder in the humandiet were required to achieve a significant decrease in plasmacholesterol. However, using fat-solubilized esterified phytosterols(e.g., the phytosterols in Benecol® margarine), it is now generallyappreciated that only 1.5-2 g of such esterified phytosterols arerequired to achieve a similar cholesterol-lowering effect. Thisdifference in potency between substantially insoluble non-esterifiedphytosterol and soluble phytosterol esters can be eliminated by heatingand fully dissolving phytosterols, and then cooling and recrystallizingthe phytosterol in the triglyceride-based medium.

Example 3 Antioxidant Effect and Chemical Stabilization of Cooking OilContaining Phytosterols

Applicants wished to determine whether admixing and dissolving asubstantial concentration of phytosterol (e.g., 10% by weight) in aheated cooking oil, would alter the chemical properties or physicalcooking properties of the cooking oil. For example, would the presenceof phytosterol accelerate the rate of oxidation or rancidity developmentin the oil, would the oil retain its original flavor, and would thecooking time for a particular food at a specified temperature beappreciably altered? Additionally it was of interest to compare theextent of oil uptake by a food fried in vegetable oil with and withoutthe phytosterol.

Accordingly, 10% by weight of the above-described soybean oil-derivedphytosterols were dissolved in a one pound quantity of heated canolaoil, and approximately 20 successive small batches of potato chips(russet potatoes, approximately 20 slices, 3-4 g per slice) were friedin each of these oils at 170° C. (338° F.) until a ratio of one pound offinished chips (1.0-1.2 g per chip) had been processed through eachpound of oil. An identical quantity of potato chips were fried in plaincanola oil as a “control”. The similarly heated spent cooking oils andthe finished potato chips were evaluated as follows: The extent ofcanola oil oxidation in each oil sample was measured using an instrumentknown as a “Foodoil Sensor” (Northern Instruments Corporation,Beachwood, Ohio) that measures the dielectric constant of the oil. Thismeasurement is a direct indicator of the relative content of peroxides,acids, and other polar compounds formed in the oil as it is beingdegraded. Following “zero-baseline” calibration of the instrument foreach unheated cooking oil formulation, the following average dielectricreadings were obtained (based upon triplicate measurements) for thepotato chip-cooked residual oils.

Plain Canola Oil 1.03 ± 0.10 Canola + 10% soybean phytosterol 0.79 ±0.15

These readings indicate that contrary to accelerating any oxidation ofthe canola oil during heating, the presence of phytosterols (10% byweight) significantly stabilized the oil against oxidation, reducing theamount of polar by-products formed in the canola oil during heating byapproximately 23%. The anti-oxidant effect and chemical mechanism thatwould explain this oil stabilization by phytosterols remains to bedetermined.

A second experiment was carried out to further characterize theantioxidant effect of phytosterols in heated cooking oils. To determinewhether a variety of heated edible fats and oils could be “stabilized”,i.e., made more resistant to oxidation in air by adding phytosterols,two different levels of soybean oil-derived phytosterols (5% and 10% byweight, and 0% as a control) were added to three different vegetableoils. Each sample of oil (5 gm) was heated in a 100 ml capacity Pyrex®glass beaker for two hours at 170° C. (338° F.). Dielectric readings ofthese oils following heating (using the same Foodoil Sensor describedabove) are provided in Table 1. As above, the dielectric reading foreach sample prior to the two hour heating in air was used as the zerobaseline reference for that sample.

A third experiment was carried out to compare the antioxidative potencyof both non-esterified sterols and stanols in heated canola oil. Thedetermination was performed under exactly the same conditions as thesecond experiment above, except that for the purpose of accelerating theoxidation rate, the heated oil temperature was increased from 170° C. to190° C. (374° F.).

Results. For each edible oil tested in the second experiment, theaddition of phytosterols significantly reduced the dielectric constantas an index of the concentration of polar compounds produced, i.e.,oxidation products formed, in the oil during heating at a temperature(170° C.) corresponding to that currently used for deep fat frying offoods. Addition of 10% by weight phytosterols to different vegetableoils resulted in approximately a 30-50% decrease in polar compoundformation during the two hour incubation (see Table 1a). This decreasewas nearly twice as great the decrease measured for the addition of 5%by weight phytosterols. This suggests that the amount of antioxidantprotection provided in edible oils and fats by phytosterols isapproximately proportional to the concentration of added phytosterols(at least for that concentration range of phytosterols tested). However,as can be seen from the results of experiment 3 (Table 1b), theeffectiveness of non-esterified phytosterols in lowering the rate ofoxidation is somewhat reduced by the higher oil temperature. Also, it isimportant to note that 10% by weight non-esterified stanols when addedto canola oil is at least twice as effective in reducing polar compoundformation during oil heating, as the same concentration ofnon-esterified sterols. The chemical explanation for this differenceremains unclear.

Before carrying out these experiments, an initial question was whethersubstantial concentrations of phytosterols (e.g., 5-10% or more) mightundesirably act as pro-oxidants during sustained heating of cooking oil.From the series of experiments herein, it is clear that theseconcentrations of phytosterols act beneficially as mild to moderateantioxidants rather than pro-oxidants.

From these results, it is believed that such phytosterols (sterols,stanols or mixtures thereof) added to edible oils and fats used inprepared food products, will also provide increased shelf-stability forthese products, via resistance to oil oxidation and ranciditydevelopment at room temperature.

Example 4 Quantitation of Cooking Oil and Phytosterol Absorbed by PotatoChips During Frying

Two other potential problems with frying foods in a phytosterol-enrichedcooking oil were examined. First, it was considered possible that theamount of fat adsorbed by fried food in a phytosterol-enriched oil mightbe greater than in regular oil. Accordingly, several tests wereconducted using individual potato slices (approximately 4 g each) thathad been pre-blotted on paper towels and deep-fried one at a time at atemperature of 180° C. in different cooking oils. Two cooking oils wereused (corn oil and canola oil) either with or without 10%soybean-derived prilled phytosterols being added and dissolved in therespective oils. Regardless of which oil was used, and regardless ofwhether phytosterols were present or absent, the average weight of thefried, drained potato chips, expressed as a percentage of the originalweight of the blotted uncooked potato slices, was constant at 31%±1%.

With regard to physically quantitating the amount of absorbed oil, ananalysis of the potato chips fried in corn oil on the one hand, and 90%by weight corn oil plus 10% (heat-solubilized) soybean-derived prilledphytosterols on the other hand, showed that the total weight proportionof oil that was solvent-extractable from the fried chips was constant,regardless of whether soy phytosterols were present or not. Morespecifically, seven potato chips (fried as described above in each ofthese two oils) were weighed, ground with anhydrous sodium sulfate, andsolvent-extracted three times with chloroform:methanol (2:1 vol/vol).This extraction method removes both phytosterols and fats from the food.The combined oil extracts were dried and weighed, and the weight ratioof extracted oil to potato chips determined. The fat content of the cornoil chips was 29±2% and the content of the phytosterol-containing chipswas 30±2%.

Second, there was a concern that the fat being adsorbed by a fried foodsuch as potato chips, might be either enriched or alternatively depletedof phytosterols, compared to the proportion of phytosterols dissolved inthe original heated cooking oil. In fact, chemical analysis of the 29%by weight cooking oil that had been extracted from the above potatochips (fried in 90% by weight canola oil plus 10% by weight soyoil-derived phytosterols, see above) showed that the extracted oilcomposition was the same as the frying oil composition (90% oil :10%phytosterol).

Phytosterol analysis employed the following method: Oil plus phytosterolcontained in potato chips was first extracted into chloroform. A portionof the chloroform (100 μl) was evaporated, redissolved in a smallquantity of isopropyl alcohol (20 μl), and then assayed using Test Kit#352 for cholesterol and other sterols (Sigma Chemical Company, St.Louis, Mo.). A test standard was prepared containing 10 micrograms ofbeta-sitosterol. This test standard essentially matched the amount ofsterol measured in the 100 micrograms of potato chip oil extract. Infact, the average value based upon five measurements from five potatochips was 9.9% by weight phytosterol. This test result indicated thatthere was no selective uptake or alternatively exclusion of thephytosterols by the potatoes as they were fried.

Therefore, given that the total weight of oil (fat plus phytosterols)adsorbed into the fried food (i.e., potato chips) appears unaffected byadded phytosterols, these phytosterols can effectively dilute andreplace a portion of the calorie-containing fat, i.e., triglycerides,that would otherwise have been adsorbed by the food during frying. Thus,a further novel benefit of using phytosterols in edible frying (orbaking) oils is to reduce the fat calorie content of the prepared food(e.g., in this Example, by approximately 10%). Without actuallyperforming the above quantitative tests, there would be no evidence thatphytosterols can substitute one for one for absorbed fat in fried food.

Concerning the amount of phytosterols provided in a one ounce serving ofpotato chips, most commercial potato chips contain at least 35% byweight vegetable oil. If this vegetable oil contains 10% by weightphytosterols, then a one ounce serving of chips would provideapproximately 1.0 g of phytosterols. At a current bulk price ofapproximately $10.00 per pound for phytosterols, the cost of thesephytosterols in a serving of potato chips would be approximately 2cents.

Example 5 Surface Oiliness of Food Fried in Phytosterol-ContainingVegetable Oil

When non-esterified phytosterols (e.g., 3% by weight or more ofphytosterols) extracted from soybeans (or tall oils) are dissolved byheating in liquid vegetable oil or fat and are subsequently cooled,their crystallization causes the oil to solidify. The degree of firmnessof the solid depends upon the phytosterol content of the oil. Forexample, when heated canola oil containing 10% by weight of dissolvedsoybean phytosterols is cooled, it solidifies to form a solid that isreminiscent of partially hydrogenated vegetable oil (PHVO). Applicantsnoticed that potato chips fried (as described above) in this oil seemedto leave less oil on ones fingers than similar chips fried in canola oilalone. Since the property of surface oiliness in fried food is generallyconsidered undesirable, an effort was made to quantitate any differencein this property among the potato chips.

A method was devised to measure the relative surface oiliness of potatochips. Potato chips fried for 1 minute at 180° C. in either canola oilor canola plus 10% by weight soybean oil-derived phytosterols werecooled for at least one hour. An assay of the total fat content of thesetwo groups of chips (method, see Example 4) showed that the formercontained 26% by weight canola oil while the latter contained 29% byweight canola oil plus phytosterols. Single potato chips were selected(weighing approximately 1.2 g each) and were gently but thoroughly wipedthree times on both sides with a single pre-weighed paper tissue(Kimwipe®, Kimberly Clark Paper Products). Each tissue was weighed on ananalytical balance before and after wiping to determine the amount ofsurface oil absorbed from the chip.

From five canola oil fried chips the following amounts of oil wereabsorbed into each tissue: 10, 7, 7, 8 and 8 milligrams. From fivesimilar chips fried in canola plus 10% by weight phytosterol thefollowing amounts of oil were absorbed: 3, 3, 3, 2 and 4 milligrams.Therefore, based upon an average of 8mg versus 3mg of surface oil, it isestimated that 10% by weight phytosterol added to a cooking oil canreduce surface oiliness of potato chips (and presumably other fried andbaked foods) approximately 2-3-fold. A similar result was obtainedcomparing potato chips fried in corn oil with chips fried in corn oilsupplemented with 10% by weight soybean oil-derived prilled sterols. Inthe latter case, an average of 10 mg of oil was absorbed from each cornoil-fried chip and only 3 mg from each corn oil plus soybean phytosterolfried chip.

Example 6 Recrystallized Non-Esterified Phytosterols in Dietary FatProvide Significant Reduction in Plasma Cholesterol Levels

The aim of this study was to determine the hypocholesterolemic efficacyof free, i.e., non-esterified phytosterols (from soybean oil) in acholesterol-responsive animal model.

Methods. Animal, diets and study design. Twelve male, 5 weeks oldCharles River Mongolian gerbils were used in the study. Gerbils wererandomly assigned to 2 groups (6 per group). Gerbils were fed for 4weeks purified diets containing 0.15% cholesterol, with 30% of caloriesprovided by fat. The overall diet contained either 0% or 0.75% ofphytosterols and 13.7% fat (detailed diet composition described in Table2). Therefore, the fat component of the diet contained either 0% or 5.5%(0.75%±13.7%) phytosterols. Phytosterols were initially heated in thefat component of the diet to allow their dissolution, and then mixedwith the other dietary components. Non-esterified sterols were allowedto freely crystallize in the fat component of the mixture as it cooled.All gerbils were given free access to water, and food was provided dailyin the predetermined amounts to meet their caloric requirement forgrowth and maintenance. Animals were housed in groups of 2-3 and werekept in a controlled environment with a 12 h light-dark cycle (light on18:00 h).

After 4 weeks of feeding of experimental diets gerbils were fastedovernight (18 h), blood samples were collected under light anesthesiawith an EDTA-wetted syringe by cardiac puncture, and afterexsanguination, livers were excised and weighted. A portion of eachliver was stored at −20° C. until analyzed. Plasma was separated fromEDTA-treated blood by centrifugation at 12,000×g for 15 min. andanalyzed within 1-2 days.

Plasma lipid analysis. Total plasma cholesterol (TC), high densitylipoprotein cholesterol (HDL-C), and triglycerides (TG) were measured byenzymatic assays (Sigma Diagnostics kits—procedures #352 for TC and #336for TG, respectively). HDL-C was assayed in the supernatant after sodiumphosphotungstate-Mg2+ precipitation of lipoproteins containingapolipoprotein B and E (Boehringer Mannheim Diagnostics, procedure543004) according to the procedure described by Weingard and Daggy(Clin. Chem. 1990, 36:575).

Results. No significant differences were observed in body weight amonggerbil treatment groups, whereas their plasma lipids variedsignificantly (Table 3).

When compared to controls, gerbils fed diets supplemented with 0.75%phytosterols and 0.15% cholesterol in the form of free phytosterols hadsomewhat smaller livers (10% less mass) that contained a dramaticallyreduced level (87-91% reduced) of esterified cholesterol (data notshown). Plasma cholesterol levels in the same gerbils were 53-57% lowerthan in the control group; HDL-C was lowered to lesser degree (23-29%)and the TC/HDL ratio was improved (decreased) significantly (by 35-40%,data not shown). Plasma triglycerides were not statistically reduced bysterol supplementation.

Discussion and Conclusion. Since non-esterified phytosterols have verylimited solubility (1.5% by weight) in dietary fat, and these sterolswere added to the dietary fat at a concentration of 5.5% by weight, most(4%±5.5% or approximately 73%) of these plant sterols were ingested inthe dietary fat as a triglyceride-recrystallized phytosterol (TRP)composition or complex. That is, the non-esterified sterols were firstdissolved in dietary fat by heating, and then cooled, resulting in theircrystallization. Therefore, it is significant and surprising that liverand plasma cholesterol-lowering results described above for thenon-esterified sterols were very favorable. More specifically, dietarysupplementation with 0.75% non-esterified sterols resulted in over 50%lower plasma cholesterol levels and approximately 90% lower livercholesterol ester levels (data not shown), with a 35% improved (lower)TC/HDL-C ratio. The results of this experiment show that thehypocholesterolemic efficacy of non-esterified sterol preparationsrecrystallized in fat is comparable to that reported in the literaturefor fat-soluble esterified sterols and stanols.

Example 7 Non-Esterified Sterols Absorbed by Potato Chips During Fryingor Dissolved and Recrystallized in Free Dietary Fat Can Reduce PlasmaCholesterol Levels

A. Gerbil Study

The aim of this study was to evaluate the hypocholesterolemic efficacyof potato chips enriched with non-esterified sterols (derived fromsoybean oil) using the same animal model system (see Example 6).

Methods. Animal, diet and study design. Fourteen male, 5 weeks oldCharles River Mongolian gerbils were used in the study. All gerbils wererandomly assigned into two groups (7 gerbils per group) and were fed for4 weeks purified diets containing 0.15% cholesterol. The detailed dietcompositions are described in Table 3. All diets contained 13.7% byweight fat, with 30% of the dietary calories being provided by the fat.Free (non-esterified) sterols were introduced into the diet at a levelof 0.75% by weight in the form of either:

Phytosterol-enriched potato chips. Potato chips were fried in canola oilthat was either supplemented or not supplemented with soybeanoil-derived phytosterols (10% by weight). When phytosterols were added,they rapidly dissolved in the oil that had been heated to 180° C. priorto frying the chips.

The control diet (see Table 4 for dietary composition) provided nosterols but contained regular commercial potato chips fried in canolaoil, to provide the same level of carbohydrate and the standard level ofdietary fat (13.7%). All other experimental conditions, including animalmaintenance, feeding, sample collecting and analytical methods were thesame as described above (see Example 6).

Hepatic cholesterol analysis. Free liver cholesterol (FC) and esterifiedliver cholesterol (EC) were determined by HPLC based on the method ofKim and Chung (Korean J. Biochem. 1984, 16:69). The free cholesterol andcholesteryl esters were separated using a Waters Radial-Pack, C18 columneluted isocratically with acetonitrile;/isopropanol (50/50 by volume) at2.0 ml/min. Absorbance of the eluate was measured at 210 nm using a UVdetector. Cholesterol concentrations (free and esterified) werecalculated by comparing the peak areas for the samples with thoseobtained for the calibration standards (Sigma Chemical Co.). Tocalculate esterified cholesterol, the sum of cholesteryl esters wasdivided by 1.67 (calculation according to Witztum et al., J. Lipid Res.1985, 26:92).

Results. The body weights of gerbils in both groups, after 4 weeks offeeding were not significantly different, whereas gerbil liver weights,liver cholesterol, and plasma lipid concentrations varied significantly(Table 5). Gerbils fed diets containing phytosterol-enriched potatochips had significantly lower liver cholesterol and plasma cholesterollevels when compared to gerbils consuming a control diet lackingsterols. Consistent with these findings, it is significant to note thatgerbils fed a diet containing regular potato chips together with thesame amount of nonesterified phytosterols that had been dissolved andrecrystallized in an equivalent quantity of canola oil, the plasma andliver cholesterol profiles were found to be very similar to those fedthe phytosterol-enriched potato chips (data not shown).

Discussion and Conclusions. The hypocholesterolemic efficacy ofnonesterified phytosterols in fortified potato chips was similar to thatobserved when phytosterols were provided in exogenous dietary fat (fullydissolving and then recrystallizing in free canola oil). The ratio offree phytosterols to fat in the standard fat level diets was0.75%/13.7%=5.5%. Applicants have shown that the solubility limit offree sterols in vegetable oil is approximately 1.5%. Therefore, aspointed out previously, most (4%+5.5%=73%) of the free phytosterol thatwas initially dissolved by heating in the dietary fat (or potato chipfat) was subsequently recrystallized to form what Applicants have termeda triglyceride-recrystallized phytosterol (TRP) composition.

In the present Example, when plant phytosterols are heated and dissolvedin a fat which is then cooled, the sterols crystallize together withtriglycerides, and the morphology, i.e., the shape and size, of thesolid material changes. At 400× magnification, large plates and extendedarrays of needle bundles of sterols associated with fat are visibleunder a microscope. These crystalline phytosterol-triglyceride solidsdiffer in their physical properties (melting temperature and crystallineappearance) from finely milled, and/or microcrystalline particlesdescribed by Tiainen et al., that have not first been dissolved andintimately combined with a triglyceride-based fat or oil.

Based upon the results of this experiment we can conclude that fryingfoods, such as potato chips, in a fat or oil supplemented with freesterols is a convenient and effective way of enriching a food withcholesterol lowering free phytosterols.

B. Human Pilot Study

Having completed the above studies in gerbils, a human pilot study wasconducted to assess the hypocholesterolemic efficacy of ingestion of afood enriched with non-esterified phytosterols. For this study, amanufacturer of tortilla chips prepared tortilla chips cooked in eithernormal fry oil or that oil containing an 8:1 ratio of fat-to-freephytosterols isolated from soybean oil. Two 1 oz bags of test chipsprovided 1.5 g of phytosterol day. The final design of the studyincluded 12 moderately cholesterolemic subjects (8 males, 4 females) andtwo test groups. Subjects initially consumed either the control chips(no sterols) or test chips (with sterols). Because the majority (n=7) ofsubjects agreed to crossover to the opposite chip after completing theirfirst 4 wk assignment, two sets of data were obtained: a straightcomparison of baseline lipid values with values after 4 wk of chips plussterols (n=10) versus a similar comparison for 9 subjects who ate thesterol-free chips (Table 6) . . . and a second, statistically strongerdirect paired-t test for the crossover data (n=7), where each person wastheir own control for the two different chips (Table 7).

In the statistically stronger comparison (Table 7) both plasmacholesterol and LDL-C, as well as the LDL/HDL ratio, declined about10-15% (clinically meaningful) without lowering beneficial HDL-C in the7 crossover subjects when consuming sterol-enriched test chips comparedto their response when eating the sterol-free chips. All 7 subjects inthe crossover group revealed a drop in LDL between 10 and 40 mg/dl. Bycontrast, no significant effect was observed for the 10 subjects duringconsumption of the control chips (non-phytosterol-containing) (Table 6).The expanded group of 9 who consumed phytosterols responded comparablyto the subgroup of 7 who also crossed over to sterol-free chips.

These data confirm that free phytosterols, when adequately dissolved andrecrystallized in fat, perform as well as phytosteryl esters in theircholesterol-lowering capacity. These results, coupled with the recentFDA allowance for a heart-healthy claim for >0.4 grams of freephytosterols per serving on such food items, indicate that this form ofphytosterol delivery is very beneficial.

Example 8 Non-Esterified Phytosterols Dissolved and SubsequentlyRecrystallized in Vegetable Oil Triglycerides Can Prevent Oil Separationin Peanut Butter

The large solubility differential between non-esterified plant sterolsdissolved in hot versus cold vegetable oil can be used advantageously informulating certain foods. As pointed out previously, most of an initialconcentration of 10% by weight plant sterols dissolved in heatedvegetable oil, e.g., potato chip frying oil, will recrystallize withtriglycerides as the oil is cooled. In the case of potato chips,crystallization of plant sterols in the oil reduces the surface oilinessof the chips. The presence of sterols in a heated vegetable oil was alsoshown to reduce the amount of polar oxidation breakdown products in thatoil as it is heated over a period of time (see Examples 3 and 10).

In the case of other high fat foods such as peanut butter, whichcontains up to 50% by weight peanut oil, between approximately 3% and 5%by weight of non-esterified plant sterols may be dissolved by heating at80-100° C. for 1-10 minutes in the peanut butter. Based upon the 50%peanut oil content, the sterols will be present in the oil portion ofthe peanut butter at a level of approximately twice the initially addedlevels, i.e., 6%-10% by weight in the oil portion. Applicants havediscovered that as little as 3% by weight non-esterified soybeanoil-derived prilled phytosterols, dissolved by heating and subsequentlycooled in peanut butter, has proven effective in partially solidifyingthe peanut oil found in a natural peanut butter. This partialsolidification prevents the natural oil separation process that isregarded as an undesirable annoyance in natural peanut butter. Thispercentage of phytosterol provides 0.9 grams sterol per 32 g serving ofpeanut butter or approximately 100% of the daily amount of plant sterolsrecommended for achieving a 10-15% reduction in the human plasmacholesterol level. This daily dose is approximately equivalent to therecommended dose of 1.3-1.5 grams of sterol esters (as provided incommercial cholesterol-reducing margarines) in which only 60% by weightof the sterol esters consists of the active sterol moiety.

Example 9 Non-Esterified Phytosterols Dissolved and SubsequentlyRecrystallized in Cocoa Butter Triglycerides can be Incorporated intoChocolate

Cocoa Butter has a melting temperature above room temperature but belowbody temperature (37° C.). This property allows chocolate, a processedfood containing approximately 30% by weight cocoa butter, to remainsolid at room temperature, and to melt in ones mouth. Soybean oilphytosterols were added to cocoa butter at a concentration of 10% byweight, and were dissolved by heating. The cocoa butter was subsequentlycooled and solidified.

A test of the softening and melting temperatures for thephytosterol-supplemented and unsupplemented cocoa butters showed thatboth were softening at approximately 30° C., and melted at approximately34° C. At 34° C., while cocoa butter became transparent,phytosterol-supplemented cocoa butter remained translucent to opaque,and exhibited a greater viscosity owing to the presence of suspendedphytosterol particles. Under the light microscope (800× magnification),the recrystallized phytosterols appeared as a fine suspension of slenderneedles and microparticules approximately 1-5 microns in width ordiameter. Thirty percent phytosterol-supplemented cocoa butter, thelatter containing 10% by weight phytosterols, was successfullyincorporated into a sweet chocolate composition. The phytosterols (3% ofthe chocolate by weight) had a negligible effect on the taste andtexture of this processed food.

Example 10 Oxidative Stabilization of Vegetable Oils Fortified withNon-Esterified Phytosterols in Production Environment

In addition to the hypocholesterolemic effect of the ingestion offat-recrystallized phytosterols, we discovered that vegetable oilsfortified with free phytosterols are substantially stabilized againstoxidation (and rancidity of stored product). This stabilization wastested in a commercial tortilla chip production setting, with analysisaccording to AOCS Recommended Practice Cd 12b-92.

The OSI measurements (each value is an average of duplicate samples,with testing carried out at 110 degrees C.) were determined by theArcher Daniels Midland (ADM) company (Decatur, Ill.) using high oleicsafflower oil samples that had been used to prepare tortilla chips. Thechips were prepared, and the oil samples harvested by the Warnock FoodCompany from heated tanks used to fry the tortilla chips. These tortillachips (prepared from standard masa flour plus 1% by weight salt) wereused in the human pilot study reported in Example 7. After frying, thetortilla chips contained 22% by weight of oil.

When phytosterols were included in the oil at a level of 12% by weight,a serving of 1 oz. (28 g) of the chips provided 22%×12%×28 g or 0.74 gphytosterols per serving. The original safflower oil (obtained fromAdams Vegetable Oil, Arbuckle, Calif.) contained 77% by weight oleicacid, 14% linoleic acid, and 8% palmitic plus stearic acids. This oilhad a stability index (OSI value) of 11.3 hours before frying wascommenced. After the frying of approximately 150 pounds of tortillachips, and maintaining the oil at a temperature of 185 degrees C. for 6hours, the OSI value of the oil had decreased to 9.5 hours.

Subsequently, fresh safflower oil and unmodified (non-esterified)soybean phytosterols (provided by ADM) were used to prepare an oil blendcontaining 88% by weight safflower oil and 12% by weight phytosterols. .This oil blend had a stability (OSI value) of 14.9 hours before fryingwas commenced. After frying approximately 132 pounds of the tortillachips, and maintaining the oil at a temperature of 185 degrees C. for 6hours, the OSI value of the oil remained essentially the same (15.1hours),

We concluded that non-esterified phytosterols exert an antioxidanteffect on a heated edible oil that carries the phytosterols, where theedible oil may be subjected to the oxidative impact of heat combinedwith air and food contact. The phytosterols, added at a level of 12% byweight, actually increased the oxidative stability of the original oilfrom 11.3 hours to 14.9 hours as measured prior to heating.

The phytosterols also reduced the loss in oxidative stability thataccompanies heating of edible oils, e.g., compare the decrease in OSIvalue from 11.3 to 9.5 hours during 6 hours of heating and frying withsafflower oil lacking phytosterols versus the OSI stability that ismaintained in the presence of phytosterols (14.9 and 15.1 hoursrespectively).

Furthermore, for application to prepared foods, we have observed thatthe shelf life (freshness) of phytosterol-fortified, fat-containingprocessed chips is extended owing to oxidative stabilization of the fat.

Unless otherwise defined herein, all terms have their ordinary meaningsas understood by one of ordinary skill in the field to which theinvention pertains. All patents and publications mentioned in thespecification are indicative of the levels of skill of those skilled inthe art to which the invention pertains. All references cited in thisdisclosure are incorporated by reference to the same extent as if eachreference had been incorporated by reference in its entiretyindividually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to obtain the ends and advantages mentioned,as well as those inherent therein. The methods, variances, andcompositions described herein as presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art, which are encompassed within the spirit of theinvention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Forexample, TRPs that are constituted using other sources of phytosterolsand/or fats and oils not listed herein, or TRPs incorporated intovarious prepared foods not listed herein, or a combination of otherphytosterol sources and other prepared foods all within the scope of thepresent invention. Thus, such additional embodiments are within thescope of the present invention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Also, unless indicated to the contrary, where various numerical valuesare provided for embodiments, additional embodiments are described bytaking any 2 different values as the endpoints of a range. Such rangesare also within the scope of the described invention.

Thus, additional embodiments are within the scope of the invention andwithin the following claims.

TABLE 1a Oxidation of heated (170° C.) oils with or withoutnon-esterified sterols (Example 3a) Increase in Dielectric Constant * 2hrs Canola oil 1.48 Canola oil + 5% Non-esterified Sterols 1.09 Conolaoil + 10% Non-esterified Sterols 0.70 Soybean oil 2.09 Soybean oil + 5%Non-esterified Sterols 1.72 Soybean oil + 10% Non-esterified Sterols1.46 Cottonseed oil 1.94 Cottonseed oil + 5% Non-esterified Sterols 1.46Cottonseed oil + 10% Non-esterified Sterols 0.85 * Oxidation of oils wasdetermined using “Foodoils Sensor” which measures the dielectricconstant of polar compounds formed in the oil during heating. Valuesindicates relative (to baseline) increases in peroxides, acids and otherpolar components formed in the oil during heating. The oils (5 g) wereheated with or without prilled phytosterols in 100 mL beakers at 170° C.

TABLE 1b Oxidation of heated (190° C.) canola oils with or withoutnon-esterified sterols or stanols (Example 3b) Increase in DielectricConstant * 2 hrs 4 hrs Canola oil 3.22 7.78 Canola oil + 10%Non-esterified Sterols 2.84 7.33 Conola oil + 10% Non-esterified Stanols2.23 6.38 * Oxidation of oils was determined using “Foodoils Sensor”which measures the dielectric constant of polar compounds formed in theoil during heating. Values indicates relative (to baseline) increases inperoxides, acids and other polar components formed in the oil duringheating. The oil (5 g) were heated with or without prilled phytosterolsin 100 mL beakers at 190° C.

TABLE 2 Composition of purified diets for gerbils (Example 6) Diet (gramper 1.0 kilo) Control (Without Non-esterified INGREDIENT % Phytosterols)Phytosterols Casein 20 200 200 Sucrose 20 200 200 Cornstarch 29.6-28.9296 289 Cellulose 10 100 100 Fat: 13.7 Coconut oil 8.1 81 81 Canola 4.343 43 Soybean oil 1.3 13 13 Mineral mix 5.0 50 50 (Ausman - Hayes)Vitamin mix 1.2 12 12 (Hayes - Cathcart) Choline chloride 0.3 3 3 FreePhytoterols 0.75 0 7.5 (prilled soybean Cholesterol 0.15 1.5 1.5premixing it with 800 mL of boiling water, to form a gel to which theremaining ingredient were added.

TABLE 3 Plasma lipids of gerbils fed for 4 weeks diets without or withnon-esterified phytosterols (Example 6). Diet Groups ControlNon-esterified (Without Phytosterols Phytosterols) (Prilled Soybean)Body weight (g) initial 53 ± 3 52 ± 2 final 66 ± 4 65 ± 3 Plasma (mg/dL)TC 153 ± 7   99 ± 9* TG  33 ± 10 24 ± 3 Values are Mean ± SD (n = 5, 6)*Significantly different (p < 0.05) from control group.

TABLE 4 Composition of purified diets for gerbils (Example 7) Diet (gramper 1.0 kilo) Chips without Chips with free phyto- phytosterolsINGREDIENT % sterolsterols (prilled soybean) Casein 20 200 200 Dextrose20 200 200 Cornstarch 10.6 106 106 Starch with chips   0-19.8 191 191Cellulose 10 100 100 Fat: 1.0-13.7 Coconut oil 62 62 Fat from chips 7567 Soybean oil 0 0 Mineral mix 5.0 50 50 (Ausman - Hayes) Vitamin mix1.2 12 12 (Hayes - Cathcart) Choline chloride 0.3 3 3 Chips prepared in268 canola oil Chips prepared in 0 268 canola oil w. 10% freephytosterols (soybean) Cholesterol 0.15 1.5 1.5 Diets were fed as gelblocks, prepared by withholding from formulation 60 g/kg of cornstarchand premixing it with 800 mL of boiling water, to form a gel to whichthe remaining ingredient were added.

TABLE 5 Plasma and liver lipids of gerbils fed for 4 weeks diets withphytosterols enriched potato chips (Example 7). Diet groups: Chips WithNon-esterified Chips Without Phytosterols Phytosterols (Prilled Soybean)Body weight(g) initial 51 ± 4 51 ± 2  final 66 ± 3 64 ± 2  Liver weight(% BW)  3.1 ± 0.1 2.8 ± 0.1* Cecum weight (% BW)  2.7 ± 0.5 2.9 ± 0.4 Adipose (Perirenal) wt (% BW)  0.32 ± 0.11 0.38 ± 10.07 Livercholesterol TC (mg/g) 39 ± 6 13 ± 4*  FC (mg/g)  5 ± 1 5 ± 0  EC (mg/g)34 ± 6 8 ± 4* Plasma TC (mg/dL) 190 ± 45 99 ± 11* TG (mg/dL) 51 ± 9 44 ±6  HDL-C (mg/dL) 68 ± 9 58 ± 9  TC/HDL-C ratio  2.9 ± 1.1 1.7 ± 0.2*Values are Mean ± SD (n = 5-7, liver cholesterol n = 4) *Significantlydifferent (p < 0.05) from control group

TABLE 6 Effect of two 1 oz bags of Tortilla chips/day, providing either1.5 g or no phytosterols, on plasma lipids in humans for 4 wk (allsubjects). Tortilla chips Chips without phytosterols Chips withphytosterols Plasma Baseline After 4 wk of chips % change Baseline After4 wk of chips % change TC (mg/dL) 226 ± 34 223 ± 32 minus 1.3 234 ± 32208 ± 30* minus 10.3 TG (mg/dL) 101 ± 52 103 ± 50 plus 2.0 117 ± 66 117± 45 0 HDL-C (mg/dL)  45 ± 11  45 ± 11 0  45 ± 10  46 ± 10 plus 2.2LDL-C (mg/dL) 161 ± 37 157 ± 35 minus 2.5 166 ± 42 141 ± 39* minus 15.1LDL/HDL-C ratio  4.3 ± 1.1  4.2 ± 0.9 minus 2.3  4.0 ± 1.5  3.3 ± 1.3*minus 17.5 Values are Mean ± SD (n = 9-10); TC = total cholesterol; TG =triglycerides; HDL-C = high-density lipoprotein cholesterol; LDL-C =low-density lipoprotein cholesterol; *Significantly lower than baseline(p < 0.05) by paired t-test.

TABLE 7 Effect of two 1 oz bags Tortilla chips, providing either 1.5 g/dor no phytosterols, on plasma lipds of humans after 4 wks (crossoverdata only). Tortilla chips without with Baseline phytosterolsphytosterols % change Plasma TC (mg/dL) 232 ± 36 228 ± 33 205 ± 34*minus 10.1 TG (mg/dL) 111 ± 52 110 ± 58 118 ± 46   plus 7.2 HDL-C(mg/dL)  48 ± 10  49 ± 10 49 ± 10 0 LDL-C (mg/dL) 162 ± 41 157 ± 38 133± 41* minus 15.3 LDL/HDL-C ratio  3.6 ± 1.3  3.4 ± 1.2  2.9 ± 1.2* minus14.7 Values are Mean ± SD (n = 7); TC = total cholesterol; TG =triglycerides; HDL-C = high-density lipoprotein cholesterol; LDL-C =low-density lipoprotein cholesterol *Significantly decrease on chipswith phytosterols (p < 0.05) by paired t-test.

1. A method for producing a fried snack food having reduced surfaceoiliness, comprising frying said snack food in a fat-based compositioncomprising at least one triglyceride-based edible oil or fat, and 2% to25% by weight of non-esterified phytosterols.
 2. The method of claim 1,wherein said fat-based composition comprises 5-10% of saidnon-esterified phytosterol.
 3. The method of claim 1, wherein saidfat-based composition comprises at least 10% of said non-esterifiedphytosterols.
 4. A method of increasing the oxidative stability of aheated frying fat composition, comprising maintaining a fat compositioncomprising at least 8% by weight non-esterified phytosterols at atemperature of at least 100 degrees C., wherein said fat composition isused for frying.
 5. The method of claim 4, wherein said fat compositionis held at said temperature for at least 1 hour.
 6. The method of claim4, wherein said fat composition is held at said temperature for at least4 hrs.
 7. The method of claim 4, wherein at least 10% by weight of saidnon-esterified phytosterols are dissolved in said fat composition. 8.The method of claim 4, wherein said fat composition oxidizes at a rateat least 20% lower than the same oil without said nonesteritiedphytosterols.